2010 Lecture 14

From Embryology

Limb Development

Lecturer : Dr Steve Palmer
Appendicular skeleton.jpg

Introduction

This lecture is an introduction to the events in limb development. Initially somites develop and then begin to differentiate forming sclerotome, dermomyotome and then dermatome and myotome. The lateral portion of the hypaxial myotome edge migrates at level of limbs (upper limb first then lower) and mixes with somatic mesoderm. Meanwhile the dermotome continues to contribute cells to myotome.

The appendicular skeleton consists of: Shoulder girdle, Upper limb (arm, hand), Pelvic girdle, Lower limb (leg, foot).

UNSW Embryology Limb Development | Limb Abnormalities 2008 Lecture 2008 Lecture | 1 slide/page | 4 slide/page | 6 slide/page

Carnegie stage 1-23

Lectopia Audio Lecture Date: 15-09-2009 Lecture Time: 12:00 Venue: BioMed E Speaker: Mark Hill Limb

Lecture Objectives

Human Embryo stage 14 SEM
  • Understanding of limb positioning
  • Understanding of differences in developmental timing of upper and lower limbs
  • Understanding of regions and factors determining limb axes
  • Understanding of limb rotation
  • Understanding of limb muscle, blood vessel, bone and nerve formation
  • Brief understanding of limb molecular factors and cell death
  • Brief understanding of limb abnormalities

Textbook References

  • The Developing Human: Clinically Oriented Embryology (8th Edition) by Keith L. Moore and T.V.N Persaud - Moore & Persaud Chapter 15 the skeletal system
  • Larsen’s Human Embryology by GC. Schoenwolf, SB. Bleyl, PR. Brauer and PH. Francis-West - Chapter 11 Limb Development


  • Before we Are Born (5th ed.) Moore and Persaud Ch16,17: p379-397, 399-405
  • Essentials of Human Embryology Larson Ch11 p207-228

Limb Buds

  • Limbs are initially undifferentiated mesenchyme (mesoderm) with an epithelial (ectoderm) covering.
  • Blood vessels then begin forming, the largest (marginal vein) is adjacent to tip of the limbbud.
  • Positioning of the limbs is distant from final location

Upper and Lower Limb

Stage20-23 limbs a.jpg

Stage14 somites limbbuds.png

Limb development occurs at different times for forelimbs and hindlimbs. In the mid-4th week, human upper limb buds first form and lower limbs about 2 days later. The limbs form at vertebra segmental levels C5-C8 (upper limbs) L3-L5 (lower limbs).


Limb Axis Formation

Four Concepts - much of the work has been carried out using the chicken and more recently the mouse model of development.

  1. Limb Initiation
  2. Proximodistal Axis
  3. Dorsoventral Axis
  4. Anteroposterior Axis

Limb Initiation

  • Fibroblast growth factor (FGF) coated beads can induce additional limb
  • FGF10 is expressed in lateral plate mesoderm prior to bud formation induces expression of FGF8 in the overlying ectoderm. FGF8 induces continued growth in the underlying mesoderm - thus a positive feedback loop
  • Anterior boundary of Hoxc6 expression coincides with the position of forelimb development


Autoregulatory loop of induction between FGF10 and FGF8

Site of FGF10 expression in the chick embryo

Positioning of the limb on the rostrocaudal (anteroposterior) axis is determined by the expression of Hox genes

Examples of Hox gene expression boundaries in the mouse Hoxb2 and Hoxb4

Limb Identity

Forelimb and hindlimb (mouse) identity appears to be regulated by T-box (Tbx) genes, which are a family of transcription factors.

  • hindlimb Tbx4 is expressed.
  • forelimb Tbx5 is expressed.
  • Tbx2 and Tbx3 are expressed in both limbs.

Related Research - PMID: 12490567 | Development 2003 Figures | Scanning electron micrographs of E9 Limb bud wild-type and Tbx5del/del A model for early stages of limb bud growth | PMID: 12736217 | Development 2003 Figures

Tbx4 expression can turn an experimentally induced forelimb into a hindlimb

Axes and Morphogens

  • Anteroposterior - (Rostrocaudal, Craniocaudal, Cephalocaudal) from the head end to opposite end of body or tail.
  • Dorsoventral - from the spinal column (back) to belly (front).
  • Proximodistal - from the tip of an appendage (distal) to where it joins the body (proximal).

Model of patterning signals in the vertebrate limb

Diffusible morphogens create a concentration gradient accross an embryonic field

Proximodistal Axis

  • Apical Ectodermal Ridge (AER) initially formed at the site of FGF10 induction
  • then AER secretes FGF8 and FGF4 slightly later
  • FGFs stimulate proliferation and outgrowth in the underlying mesenchyme

apical ectodermal ridge | AER and vascular channel

Fibroblast growth factors (FGF)

  • 22 FGF genes identified in humans
  • bind membrane tyrosine kinase receptors
  • Patterning switch with many different roles in different tissues

FGF receptors

  • comprise a family of at least 4 related but individually distinct tyrosine kinase receptors (FGFR1- 4) similar protein structure
  • 3 immunoglobulin-like domains in extracellular region
  • single membrane spanning segment
  • cytoplasmic tyrosine kinase domain

FGF receptors are paired proteins on the cell surface with an internal tyrosine kinase domain

Dorsoventral Axis

  • Ventral muscles - flexors Dorsal muscles - extensors
  • Early grafting experiments showed that the D/V signalling centre resided in the dorsal ectoderm
  • Wnt7a is a diffusible morphogen that is secreted by dorsal ectoderm cells
  • Wnt7a induces the expression of the homeobox gene Lmx1 in the underlying mesoderm adjacent to the dorsal surface
  • The homeobox gene Engrailed (En1) is expressed in the opposite ventral ectoderm

Consequence of Wnt7a deficiency in the mouse forelimb

Wnt7a

  • name was derived from 'wingless' and 'int’
  • Wnt gene first defined as a protooncogene, int1
  • Humans have at least 4 Wnt genes
  • Wnt7a gene is at 3p25 encoding a 349aa secreted glycoprotein
  • patterning switch with different roles in different tissues
  • One WNT receptor is called Frizzled (FZD) - named after a drosophila phenotype
  • Frizzled gene family encodes a 7 transmembrane receptor

Anteroposterior Axis

  • Zone of polarizing activity (ZPA)
  • a mesenchymal posterior region of limb
  • secretes sonic hedgehog (SHH)

ZPA secretes SHH and determines the anteroposterior axis of the limb bud

Dynamic development - time, the 4th dimension

  • Different Hox genes are expressed at different times in the developing limb bud and pattern the fine structure of the limb.
  • Structures are determined in a proximal>distal direction with time, i.e. proximal structures such as the humerus bone are laid down first.

Hox genes and dynamic patterning of the limb

Cellular origins of the limb

Limb cartilage and bone

  • Derived from local proliferating mesenchyme derived from the somatic lateral plate mesoderm (somatopleure)

Limb muscle and dermis

  • Skeletal muscle derived from somites, the hypaxial part of the myotome
  • Pax3 positive migratory myoblasts invade the limb bud
  • Similarly, dermal cells also invade derived from the dermomyotome
  • Both maintain the identity of the somite from which they were derived so that innervation corresponds to the same spinal nerve root.
  • Note that dermatomes are rotated due to embryonic limb rotations

Origin of limb muscle cells - Migrations traced by grafting cells from a quail embryo into a chick embryo

  • two species very similar in development
  • quail cells recognizable by distinctive nucleoli
  • Quail somite cells substituted for somite cells of 2 day chick embryo
  • wing of chick sectioned a week later
  • found muscle cells in chick wing derive from transplanted quail somites

Dorsal/Ventral Muscle Mass

Forelimb Muscles

Limb Muscle - Differentiation, Skeletal muscle differentiates the same

  1. Muscle precursor cells migrate to the muscle location
  2. Form beds of proliferating myoblasts
  3. Myoblasts fuse together to form myotubes
  4. Myotubes begin to express contractile proteins, form sarcomeres
  5. mature into myofibers, Innervation determines final muscle maturation

Dermomyotome MyoD

Hand and Footplates

Depletion of BMP Signaling Causes Interdigital Syndactyly
  • 5th week- hand and footplates appear at the ends of limb buds and ridges form digital rays
  • Cells between the digital rays are removed by programmed cell death (apoptosis)
  • 3-5 day difference between hand and foot development


hand growth

Apoptosis

Cell Biology - Cell Death Lecture | Cell Biology - Apoptosis Lecture

Fluorescent staining of cells undergoing apoptosis in the limb

Limb Rotation

Stage20-23 limbs a.jpg
  • 8th week limbs rotate in different directions (Humans Stage 20-23)
  • thumb and toe rostral
  • knee and elbow face outward
  • upper limb rotates dorsally
  • lower limb rotates ventrally

Limb Innervation

brachial plexus
Adult Dermatomes
  • spinal cord segmental nerves form a plexus adjacent to each limb
  • Brachial (upper) lumbar (lower)
  • Plexus forms as nerves invade the limb bud mesechyme
  • Fetal period - touch pads become visible on hands and feet

brachial plexus origin

Limb Abnormalities

Congenital Hip Dislocation

Congenital Hip Dislocation
  • Instability: 1:60 at birth; 1:240 at 1 wk: Dislocation untreated; 1:700
  • congenital instability of hip, later dislocates by muscle pulls or gravity
  • familial predisposition female predominance
  • Growth of femoral head, acetabulum and innominate bone are delayed until the femoral head fits firmly into the acetabulum

limb abnormalities

Maternal

  • thalidomide Phocomelia
  • short ill-formed upper or lower limbs
  • hyperthermia

Genetic

  • Trisomy 21 - Downs syndrome
    Trisomy21 hand.jpg
  • Human Gene Mutations - mutation of any of the patterning genes will result in limb abnormalities

Type II syndactyly- HoxD13

Muscle Development

Duchenne Muscular Dystrophy

  • X-linked dystrophy
  • large gene encoding cytoskeletal protein- Dystrophin
  • progressive wasting of muscle, die late teens

Becker Muscular Dystrophy

  • milder form, adult onset


Online Links

References

Textbooks

  • The Developing Human: Clinically Oriented Embryology (8th Edition) by Keith L. Moore and T.V.N Persaud - Moore & Persaud
  • Larsen’s Human Embryology by GC. Schoenwolf, SB. Bleyl, PR. Brauer and PH. Francis-West -

Online Textbooks

  • Madame Curie Bioscience Database Chapters taken from the Madame Curie Bioscience Database (formerly, Eurekah Bioscience Database)

Search

Images

Stage13

Stage13 bf1c.jpg Stage13 sem1c.jpg

Stage14

Stage14 bf2cl.jpg Stage14 sem1c.jpg


Glossary Links

Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | Numbers | Symbols | Term Link

Course Content 2010

Embryology Introduction | Cell Division/Fertilization | Lab 1 | Week 1&2 Development | Week 3 Development | Lab 2 | Mesoderm Development | Ectoderm, Early Neural, Neural Crest | Lab 3 | Early Vascular Development | Placenta | Lab 4 | Endoderm, Early Gastrointestinal | Respiratory Development | Lab 5 | Head Development | Neural Crest Development | Lab 6 | Musculoskeletal Development | Limb Development | Lab 7 | Kidney | Genital | Lab 8 | Sensory | Stem Cells | Stem Cells | Endocrine | Lab 10 | Late Vascular Development | Integumentary | Lab 11 | Birth, Postnatal | Revision | Lab 12 | Lecture Audio | Course Timetable


Cite this page: Hill, M.A. (2018, December 10) Embryology 2010 Lecture 14. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/2010_Lecture_14

What Links Here?
© Dr Mark Hill 2018, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G